The intestinal mucosa exists in a functional equilibrium with the complex lumenal milieu which is dominated by a wide variety of commensal microbial species. Despite the continuous confrontation with bacterial products the normal mucosa exists in a state of minimal immune activation but must promptly mount an effective defense typically dependent on activation of inflammatory pathway when infection with a pathogen occurs. The overall goal of the present proposal is to define the mechanisms through which the epithelium interfaces with normal and pathogenic lumenal bacteria and the processes determining the functional outcome of those interactions whether normal homeostasis or active inflammation. These studies are based on the presumption that a family of pattern recognition receptor designated toll-like receptors (TLRs) found to be present on intestinal epithelial cells allow the mucosa to sample the bacterial milieu and play a determining role in the functional outcome of that interaction. The proposed studies address two interrelated hypotheses: (1) TLRs play a critical role in maintaining the integrity of epithelial/mucosal barrier function, (2) functional cellular responses reflect the balance between positive and negative regulatory signaling molecules. Efforts to achieve the broad aims of this proposal and test these hypotheses will be pursued through studies of three specific aims: Specific Aim I: To delineate the mechanisms enhancing intestinal epithelial integrity mediated through TLR activation Specific Aim II: To define mechanisms of positive regulation of TLR function in IEC: role of AKAP13. a novel guanine exchange factor. Specific Aim III: To define mechanisms of negative regulation of TLR function in IEC: role of a novel inhibitor Tribbles2 (TRB2) Exploration of the relevance of insights gained through in vitro characterization by studies in in vivo models should advance understanding of the role of TLRs in inflammatory bowel disease. Collectively these studies should provide new insights into the role of the TLRs, key elements of the innate immune system, in modulating the dynamic balance between controlled surveillance and appropriate response to mucosal challenge by lumenal flora.